This invention relates to a device and method for the isolation of macromolecules (including proteins and nucleic acids) from a gel or other polymer supports (or also in solution) to a predetermined volume (10 ul to one ml or more). Furthermore, the macromolecule can be dialyzed and concentrated in the same device.
Electrophoresis traditionally involves establishing an electric field between opposite ends of a solid gel matrix made of agarose and/or polyacrylamide, cellulose acetate. The sample (usually a protein or nucleic acid) is applied on one end of the gel and subjected to electrophoresis. Depending upon various physicalchemical characteristics, the sample migrates through the electrical field at a particular velocity. After electrophoresis, the gel is removed, stained with reagents specific for proteins and/or nucleic acids, destained with organic solvent mixtures and photographed. Whereas electrophoretic separation of macromolecules is an established technique, the elution of macromolecules from the gel is a difficult nonreproducible procedure. Such macromolecules are of potential commercial value because of their applications in science and medicine. Over the last decade or so, various attempts have been made to sluts such macromolecules from gels.
Perhaps the simplest procedure for the elution of macromolecules involves washing the gel and vigorously mixing it with a solution containing salt for several hours. Next, the entire mixture is centrifuged. The macromolecule eluted with the salt solution is precipitated with ethanol. This procedure is very time-consuming and the recovery of macromolecules varies considerably depending upon the nature of the macromolecule. Moreover, ethanol precipitation results in denaturation of several proteins or loss of biological activity of macromolecules.
A similar approach has been taken to elute nucleic acids from agarose gels. Here, agarose gel is melted by heating to 65.degree. C. The mixture is extracted with phenol and the samples eluted. As expected, recoveries are usually low with this procedure. In addition, phenol is a highly toxic and biohazardous substance.
Since diethylaminoethyl(DEAE) cellulose binds deoxyribonucleic acid (DNA), it has been employed to elute DNA from gels. The procedure involves i) electrophoretic transfer of DNA from gels to DEAE-paper. ii) alternatively, DEAE-paper is inserted into slots immediately under each band, thus DNA is transferred electrophoretically. Although these procedures yield excellent recoveries, they are highly dependent on technique and the apparatus is expensive.
Decomposing the gel with chemicals, followed by trapping the macromolecules on glass beads and their elution with salt solution is another method of elution. However, this method is dependent on buffer conditions.
In 1985, Kartenbech introduced an electroelution apparatus (U.S. Pat. No. 4,552,640). This apparatus consists of an upper electrode in the upper chamber and the lower chamber to hold buffer solution and a lower electrode. The upper chamber is separated from the lower chamber by a septum, and the two chambers are connected by a connecting passage within the septum. The end of the lower chamber holds a dialysis membrane, wherein the electrophoretically eluted protein or polypeptide is collected. There are several disadvantages with this apparatus. These are: i) since the volume of the lower chamber is large, it results in dilution of the sample, and ii) since the surface area of the dialysis membrane is large it results in non-specific adsorption of macromolecules resulting in very low recoveries.
In 1985, Walsh introduced an apparatus to sluts nucleic acids (U.S. Pat. No. 4,545,888). This apparatus has features to introduce multiple copies of transfer chamber, filter discs to hold DEAE cellulose and negative electrode. Basically, in this procedure the sample is electrophoresed and collected on DEAE resin (held by a filter disc) at the bottom end of the lower chamber. Next, the filter disc is removed and DNA eluted from the resin employing standard elution protocols. This procedure requires an additional step involving the slution of nucleic acids from DEAE. Moreover, its application to elute proteins and polypeptides is uncertain.
In 1987, Burd introduced an electroelution method and apparatus (U.S. Pat. No. 4,699,706). This apparatus has features in which the electroeluted sample passes through a glass frit and is collected in a semipermeable membrane at the bottom end of the lower chamber. Some of the disadvantages of this apparatus are: i) the dialysis membrane must be held in place by a retaining ring, a gasket and internal shoulders built in the equipment. There are several disadvantages with this equipment. For example, i) this is a rather complex setup and the success depends upon the technique, ii) because the dialysis membrane is smaller then the diameter of the glass frit, it results in poor recovery, iii) use of dialysis membrane results in non specific adsorption of macromolecules, which also contributes to low recovery. iv) there is no possibility of capping the columns to harvest the sample collected in the membrane, v) when the sample cup is removed, it leads to the disruption of the sample collected as it leaks through the filter disc and/or fluid held in the sleeve holding the cup.
In 1986, Clad introduced an apparatus for electroeluting macromolecules from gel (U.S Pat. No. 4,608,147). This apparatus contains an upper chamber which holds a permeable membrane (pore size about 0.2 micro meter) through which macromolecules can migrate downstream. The sample is collected in the lower chamber on top of an impermeable membrane having a molecular weight greater than 1000. Following elution, the polarity of the electric field is reversed for 10 to 15 seconds, so that the macromolecules adsorbed to the inner surface of the outer membrane are released from the membrane into the trap space. There are several disadvantages of this apparatus. These are: i) the use of an impermeable membrane in the lower chamber results in dilution of sample, thus requiring concentration further, ii) because the sample is contaminated with the electrophoretic buffer, an additional step (e.g. dialysis) is required to remove such contaminants.
In 1990, Brautigam and Gorman introduced an electroelution apparatus (U.S. Pat. No. 4,964,961). This equipment consists of a tapered tube divided by a porous disc into an open upper section and a lower section which can be closed by a removable cap. The equipment has a dialysis membrane equal to the diameter of the removable cap and is affixed to it to close off the lower section. After electroelution, the upper section is closed. The sample is collected through the cup and dialysis membrane at the bottom end of the tube. The disadvantages of this equipment are: i) the sample is contaminated and diluted with the electrophoretic buffer; accordingly, it requires dialysis and concentration, further adding to the time effort for such procedures, and ii) non-specific adsorption of sample to the dialysis membrane results in loss of recovery.
In summary, at the present time the separation of macromolecules from a solid phase matrix involves procedures that are elaborate, labor-intensive, expensive, and yields are poor. Such equipment does not have dialysis capabilities resulting in considerable loss of precious macromolecules. In addition, the handling of samples subjects the investigator to a higher chance of contamination with macromolecules. For example, the elution of radiolabeled macromolecules, toxins, and viruses involves electrophoresis resulting in contamination of the apparatus with unbound radioactive material. The eluted protein is usually repeatedly centrifuged against a membrane filter or precipitate in a concentrated salt solution. The resulting material is next dialyzed in a bag which results in removal of salts and at the same time increases the sample volume. Such a sample is next concentrated by ultrafiltration. Taken together, all of these procedures are time-consuming, and often the yields are low (10-30%) compared to the starting material. Furthermore, dialysis and concentration (if required) of the sample are additive steps required to prepare samples, which result in further low recoveries.